In recent years, an increasing number of formulations of pharmacologically active proteins or peptides have been developed for commercial use. However, such drugs usually have a short half-life in the blood and most of them are injections that must be administered repeatedly at frequent intervals, thus imposing excessive burdens on patients during drug administration. Hence, there is a demand for practical sustained-release formulations of protein or peptide drugs, which exert their efficacy in as small amounts as possible and which permit reduced frequency of administration.
Sustained-release formulations of pharmacologically active proteins or peptides will cause denaturation or aggregation of the proteins or peptides during formulation preparation or sustained release, which results in a reduced recovery rate and constitutes a major obstacle to their development for commercial use. It has been attempted to prepare sustained-release formulations based on a biodegradable polymer matrix such as polylactic acid-polyglycolic acid copolymer (PLGA), but such formulations have been reported to cause protein denaturation and/or aggregation due to the hydrophobicity of the matrix, a drying step and/or a decrease in pH (see J. Pharm. Sci. vol. 88, pp. 166-173, 1999 and J. Microencapsulation vol. 15, pp. 699-713, 1998). On the other hand, there are also reports of sustained-release formulations based on a hydrophilic hydrogel matrix with reduced risks of these problems, but such formulations are not ready for commercial use. In terms of safety, a material used as a sustained-release matrix should combine non-antigenicity, non-mutagenicity, non-toxicity and biodegradability. Thus, no sustained-release formulation is now ready for commercial use in all aspects, i.e., encapsulation rate and recovery rate of proteins or peptides, as well as safety.
Hyaluronic acid (HA), a biomaterial (polysaccharide) isolated from the vitreous body of bovine eyes in 1934 by K. Meyer, has been known as a major component of extracellular matrix for a long time. HA is a kind of glycosaminoglycan composed of disaccharide units in which D-glucuronic acid and N-acetylglucosamine are linked to one another via β(1→3)glycosidic linkages (Formula (VI)).

There is no species difference in the chemical and physical structure of HA and humans also have a metabolic system for HA; HA is therefore the safest medical biomaterial in terms of immunogenicity and toxicity. Recently, microbial mass production of high-molecular-weight HA became possible allowing commercial use of HA in the fields of therapeutic agents for degenerated cartilage, cosmetics, etc.
There are also many reports on crosslinking techniques using hyaluronic acid as a matrix and sustained release of protein or peptide drugs from hyaluronic acid gels. Techniques known for gelling HA via chemical crosslinking include the carbodiimide method (see International Publication No. WO94/02517), the divinylsulfone method (see JP 61-138601 A), and the glycidyl ether method (see JP 05-140201 A). In general, when a protein or peptide is introduced into a crosslinked gel for encapsulation purposes, it results in a low introduction rate because of problems arising from compatibility and electrostatic repulsion between a hyaluronic acid derivative and the protein or peptide. In contrast, when in situ crosslinking is performed in the presence of a protein or peptide, it is advantageous in that the protein or peptide can be held in a gel at a high encapsulation rate. There are some reports showing that such in situ crosslinking is adapted for encapsulation of proteins or peptides into hyaluronic acid derivative gels to give sustained-release formulations (see, e.g., U.S. Pat. No. 5,827,937). However, there arises a problem of recovery rate when such an approach is used for in situ crosslinking of hyaluronic acid in the presence of proteins or peptides. As an example, a method is reported in which a hyaluronic acid derivative (HA-HZ) modified to have a hydrazide group (HZ) is crosslinked with a crosslinking agent comprising N-hydroxysuccinimide (NHS) (see International Publication No. WO95/15168). This method is intended for in situ crosslinking under physiological conditions and limits crosslinkage formation at pH 7.4 to pH 8.5. However, the inventors'investigations have shown that this method results in low recovery rates of proteins or peptides from the thus obtained hyaluronic acid derivative gel. This is because the proteins or peptides are partially reacted (mainly at their amino groups) with the crosslinking agent during crosslinking reaction resulting in crosslinked proteins. This method also suffers from a problem in that denatured proteins or peptides remaining in the gel have reduced biological activity and, if anything, are responsible for the development of antigenicity. It is an essential requirement for pharmaceutical preparations that the encapsulated drug is released at a high recovery rate, and no method is known for chemically crosslinking and gelling hyaluronic acid without causing proteins or peptides to react. Also, another method has been reported to encapsulate proteins or peptides at high recovery rates, in which polyethylene glycol (PEG) is used as a matrix and crosslinked through nucleophilic addition reaction of unsaturated functional groups (see International Publication No. WO00/44808), but this method suffers from a problem in that fragments of non-biodegradable polyethylene glycol remain in the resulting material.